Bacillus pumilus, microbial inoculant, method and use

Abstract

The present application belongs to the technical field of microorganisms, and particularly relates to a bacillus pumilus, a microbial agent, a method and application. The present application provides a bacillus pumilus G15 with a preservation number of CGMCC No.32839. The bacillus pumilus G15 of the present application has a significant inhibitory effect on a variety of pectobacterium FN20111. The present application further provides a microbial agent. Potting prevention effect determination shows that the bacillus pumilus G15 has a high prevention effect on the variety of pectobacterium FN20111.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus pumilus, a bacterial agent, a method, and its application. Background Technology

[0002] Potato blackleg is caused by *Pectobacterium* spp. It is a relatively common bacterial disease in potato production. Aerial stalk rot, on the other hand, is a new disease caused by three new species of the same genus as the pathogen that causes blackleg: *P. parvum*, *P. versatile*, and *P. brasiliense*.

[0003] Aerial stalk rot of potatoes is a bacterial disease that primarily affects vigorous varieties such as Hisense 6, 226, and Wotu 5. Under certain conditions, V7 can also break out. The disease mainly results from bacterial infection after damage to the stem base, causing blackening and stickiness. If not controlled promptly, it can lead to widespread infection throughout the field, severely impacting potato yield and quality. Controlling aerial stalk rot is more difficult than controlling late blight. Because aerial stalk rot has only become prevalent in recent years, its disease development patterns, seed potato carrier status, and effective control methods are still underdeveloped. Currently, no strains have been found that offer good control against both potato aerial stalk rot and potato blackleg. Summary of the Invention

[0004] In view of this, the present invention provides a strain of Bacillus pumilus, an inoculum agent, a method, and an application. The Bacillus pumilus provided by the present invention can control potato aerial stem rot and potato black shank.

[0005] To address the aforementioned technical problems, the present invention proposes the following technical solution:

[0006] This invention provides a strain of Bacillus pumilus G15, with accession number CGMCC No. 32839.

[0007] This invention provides a microbial agent comprising Bacillus pumilus G15 as described in the above technical solution.

[0008] Preferably, the effective viable count of Bacillus pumilus G15 in the bacterial agent is ≥1×10⁻⁶. 7 CFU / mL.

[0009] Preferably, the application forms of the Bacillus pumilus G15 include bacterial suspension and / or fermentation broth.

[0010] Preferably, the preparation of the fermentation broth includes: inoculating the Bacillus pumilus G15 into a culture medium for cultivation to obtain the fermentation broth;

[0011] The preparation of the bacterial suspension includes: centrifuging and resuspending the fermentation broth to obtain the bacterial suspension.

[0012] This invention provides the application of Bacillus pumilus G15 or the bacterial agent described in the above-mentioned technical solution in the prevention and control of pathogens of the genus Pectobacterium spp.

[0013] This invention provides the application of Bacillus pumilus G15 or the inoculant described in the above-mentioned technical solution in the prevention and control of potato diseases.

[0014] Preferably, the diseases include potato blackleg and / or potato aerial stem rot.

[0015] This invention provides a method for preventing and controlling potato diseases, which involves applying the Bacillus pumilus G15 or the inoculant described in the above-mentioned technical solution to potatoes.

[0016] Preferably, when used in the field, the application rate of the microbial agent is 1.9–2.1 L / 667 m³. 2 When used in potted plants, the amount of the microbial agent sprayed is 28-32 mL per seedling.

[0017] The beneficial effects of this invention are as follows: This invention provides a strain of *Bacillus pumilus* G15, which exhibits a significant inhibitory effect on *Bacillus multiflora* FN20111. After treatment with G15 against FN20111, the weight of the rotten portion of potatoes was 0.23 ± 0.04 g, indicating a reduction in the weight of the rotten portion. This invention also provides a microbial agent; pot experiment results show that *Bacillus pumilus* G15 has a control efficacy of 52.87% against *Bacillus multiflora* FN20111. Attached Figure Description

[0018] Figure 1 The colony morphology and Gram staining of Bacillus pumilus G15 are shown.

[0019] Figure 2 The results of screening for the biocontrol strain of Bacillus pumilus to inhibit the aerial stem rot pathogen are shown. The left side shows the inhibition effect of G15 on the multi-pectinobacter FN20111, and the right side shows the water control group.

[0020] Figure 3 A phylogenetic tree of closely related species of Bacillus pumilus G15 constructed based on the maximum likelihood method;

[0021] Figure 4Phylogenetic trees constructed for strains FN20211 and FN20111 based on the gapA gene sequence;

[0022] Figure 5 To illustrate the virulence of P. versatile and Bacillus pumilus G15 on potato tubers, A shows longitudinal sections of tubers inoculated with P. versatile FN20111 and Bacillus pumilus G15; B shows the difference in internal decay weight of tubers under different treatments.

[0023] Figure 6 The graph shows the efficacy of Bacillus pumilus G15 against P. versatile in potato tubers. In this graph, A shows the effect of strain G15 on FN20111 (P. versatile) in detached tubers; B shows the difference in internal decay weight of tubers under different treatments; P. versatile represents strain FN20111.

[0024] Figure 7 The image shows the pot control efficacy of Bacillus pumilus G15 against P. versatile FN20111. In this image, A is a longitudinal section of potato stems under different treatments; B is an analysis of the differences in the length of internal rot in the stems under different treatments; and P. versatile represents strain FN20111.

[0025] Biological Preservation Instructions

[0026] Bacillus pumilus G15 was deposited on November 28, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32839. Detailed Implementation

[0027] This invention provides a strain of Bacillus pumilus G15, with accession number CGMCC No. 32839.

[0028] The Bacillus pumilus G15 strain of the present invention was obtained by screening rhizosphere soil samples. The colonies of Bacillus pumilus G15 on LB agar plates are milky white, uniformly stained, and without protrusions. The gyrB gene sequence is shown in SEQ ID No. 1. Based on the maximum likelihood method, strain G15 was identified as Bacillus pumilus.

[0029] SEQ ID No.1:ATGCGTTATCTACGACYTTAGACGTGACCGTATATCGTG。

[0030] The potato tuber and pot experiments in the embodiments of the present invention demonstrate that Bacillus pumilus G15 has a significant inhibitory effect on Bacillus multiflora FN20111, and can prevent and control potato aerial stem rot and potato black shank disease.

[0031] This invention provides a microbial agent comprising Bacillus pumilus G15 as described in the above technical solution.

[0032] In this invention, the effective viable count of Bacillus pumilus G15 in the bacterial agent is preferably ≥1×10⁻⁶. 7 CFU / mL, more preferably 1×10⁻⁶ 7 CFU / mL.

[0033] In this invention, the preferred application forms of the Bacillus pumilus G15 include resuspension and / or fermentation broth.

[0034] In this invention, the preparation of the fermentation broth preferably includes: inoculating the Bacillus pumilus G15 into a culture medium for cultivation to obtain the fermentation broth.

[0035] In this invention, the *Bacillus pumilus* G15 is inoculated into a solid culture medium for activation culture to obtain single colonies of G15. The preferred inoculation method is streak inoculation. There are no special limitations on the parameters of the streak inoculation; conventional methods are acceptable. The activation culture temperature is preferably 30℃~37℃, more preferably 33~37℃; in specific embodiments of this invention, the activation culture temperature is preferably 30, 32, 33, 34, 35, 36, or 37℃. The activation culture time is preferably 21~26 hours, more preferably 23~24 hours; in specific embodiments of this invention, the activation culture time is preferably 21, 22, 23, 24, 25, or 26 hours. The activation culture is preferably completed under static conditions. The solid culture medium preferred in this invention includes LB solid culture medium.

[0036] In this invention, the obtained G15 single colony is preferably inoculated into a liquid culture medium for seed culture to obtain a seed solution. This invention does not have a specific limitation on the inoculation method; conventional methods are acceptable. The seed culture temperature is preferably 33℃~38℃, more preferably 36~37℃; in specific embodiments of this invention, the seed culture temperature is preferably 33, 34, 35, 36, 37, or 38℃. The seed culture time is preferably 12~19h, more preferably 12~16h; in specific embodiments of this invention, the seed culture time is preferably 12, 13, 14, 15, 16, 17, 18, or 19h. The seed culture rotation speed is preferably 170~220rpm, more preferably 180~200rpm. The liquid culture medium of this invention preferably includes LB liquid culture medium.

[0037] In this invention, the seed culture is preferably transferred to a liquid culture medium for fermentation to obtain a fermentation broth. The fermentation temperature is preferably 31℃~37℃, more preferably 35~37℃; in specific embodiments of this invention, the fermentation temperature is 31, 34, 35, 36, or 37℃. The volume of the seed culture transferred is preferably 1%~7% of the culture medium volume, more preferably 2%~5%. This invention preferably involves fermentation until the OD of the culture broth reaches a certain value. 600 The value is 0.8 to 1.0. The preferred rotation speed for cultivation according to this invention is 190 to 210 rpm, more preferably 195 to 200 rpm.

[0038] In this invention, the preparation of the resuspension preferably includes: centrifuging and resuspending the fermentation broth to obtain a bacterial suspension.

[0039] In this invention, the fermentation broth is preferably centrifuged to obtain G15 cells, which are then washed and resuspended to obtain a resuspension. The washing and resuspension solutions used in this invention are preferably 10 mM NaCl solution or physiological saline. This invention does not have a specific limitation on the number of washing cycles; conventional methods are sufficient.

[0040] The bacterial agent and Bacillus pumilus G15 described in this invention can be used alone or in combination with other Bacillus species.

[0041] This invention provides the application of Bacillus pumilus G15 or the bacterial agent described in the above-mentioned technical solution in the prevention and control of pathogens of the genus Pectobacterium spp.

[0042] The pathogenic bacteria of the genus Pectobacterium spp. described in this invention preferably include P. versatile.

[0043] This invention provides the application of Bacillus pumilus G15 or the inoculant described in the above-mentioned technical solution in the prevention and control of potato diseases.

[0044] The pathogens of the diseases described in this invention preferably include the genus *Pectobacterium* spp. The genus *Pectobacterium* spp. described in this invention preferably includes *P. versatile*.

[0045] The diseases described in this invention preferably include potato black shank and / or potato aerial stem rot. *Pectobacterium versatile* is the pathogen of potato black shank and potato aerial stem rot. The fact that *Bacillus pumilus* G15 can control pathogens of the genus *Pectobacterium* spp. indicates that it can control potato black shank and potato aerial stem rot.

[0046] The preferred method of application of the present invention includes: applying the Bacillus pumilus G15 or the inoculant described in the above technical solution to potatoes.

[0047] This invention provides a method for preventing and controlling potato diseases, which involves applying the above-described technical solution or the above-described technical solution's Bacillus pumilus G15 or the above-described inoculant to potatoes.

[0048] The microbial agent of the present invention can be applied in the field or in pots. The specific application amounts for the field and pots are as follows: When used in the field, the preferred application method includes spraying; the preferred application amount for spraying is 1.9–2.1 L / 667 m³. 2 More preferably 2L / 667m 2 The preferred time for application is during the budding stage of potato plants, and the preferred number of applications is 6 times. The preferred time for the first application is during the budding stage of potato plants, and the preferred interval between two applications is 10 days.

[0049] When used in potted plants, the preferred dosage of the microbial agent is 28-32 mL per potato plant, more preferably 30 mL; the preferred method of application is spraying, the preferred spraying site is the potato stem, the preferred number of applications is once, and the preferred spraying time is about 30 days after the potato seedlings emerge from the pot.

[0050] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1: Isolation and purification of Bacillus strains from rhizosphere soil

[0052] Using the soil dilution method, 10g of rhizosphere soil was weighed and added to an Erlenmeyer flask containing 90mL of sterile water. The flask was shaken at 200rpm / min for 30min at 28℃, and the soil suspension was then collected. 10mL of the soil suspension was added to 90mL of 85% physiological saline, and the mixture was incubated in a water bath at 80℃ for 15min, followed by serial dilution to 100%. -3 10 -4 10 -5 100 μL of each gradient was spread onto blank LB medium, with each gradient treatment repeated three times. The mixture was then incubated at 37°C for 24 h. Single colonies were picked and streaked for purification, yielding multiple bacterial strains. Colonies with surface wrinkles were designated as strain G15.

[0053] A single colony of the bacteria-free strain G15 was used to prepare a seed culture, which was then mixed with glycerol at a volume ratio of 1:1 and stored at -80°C. The glycerol concentration was 50%.

[0054] The bacterial strain was streaked onto blank LB medium, and the colony color, edge, and morphological characteristics were observed under a light microscope. The bacterial suspension was stained with Gram stain, and the color and shape of the bacterial cells were observed under a light microscope. The results are shown in the figure. Figure 1 . Figure 1 The images show the colony morphology and staining of Bacillus pumilus G15.

[0055] Strain G15 appears milky white on LB plates, with uniform staining and no raised areas (see...). Figure 1 Gram-positive bacteria.

[0056] Example 2

[0057] 1. Materials and Methods

[0058] (1) Test strains

[0059] The pathogens causing potato aerial stem rot were provided by the Potato Diseases and Plant Pathogens Innovation Team of the College of Plant Protection, Hebei Agricultural University. The pathogens causing potato aerial stem rot are Pectobacterium parvum FN20211 and Pectobacterium versatile FN20111. For Pectobacterium parvum FN20211, please refer to "Wang Jinhui, Han Wanxin, Pirhonen Minna, Pan Yang, Zhang Dai, Zhao Dongmei, Li Qian, Cheng Jianing, Yang Zhihui, Zhu Jiehua. High-Quality Complete Genome Resource of Pectobacterium parvum Isolate FN20211 Causing Aerial Stem Rot of Potato.[J].Molecular plant-microbe interactions:MPMI,2022,35(6):MPMI01220009A-MPMI01220009A.";

[0060] For Pectobacterium versatile FN20111, see "Han Wanxin, Wang Jinhui, Li Zheng, Pan Yang, Zhang Dai, Zhao Dongmei, Li Qian, Yang Zhihui, Zhu Jiehua. First Report of Pectobacterium versatile Causing Aerial Stem Rot of Potato in China. [J]. Plantdisease, 2021."

[0061] (2) Test culture medium

[0062] The activation, preservation and propagation of pathogens and Bacillus were carried out using Luria-Bertani (LB) medium. The specific formula for LB liquid medium is as follows: 10g peptone, 5g yeast extract, 10g sodium chloride, distilled water to a final volume of 1L, pH adjusted to 7.0, and 20g agar powder added per liter of LB solid medium.

[0063] 2. Screening of biocontrol strains - Plate inhibition test

[0064] *Pectinobacterium granulosum* FN20211 and *Pectinobacterium diversicolor* FN20111 were activated on LB agar plates. Single colonies were picked and added to LB liquid medium and cultured at 28°C with shaking at 200 rpm until OD200. 600 With a value of 0.8, we obtained culture media of *Pectinobacterium granulosum* FN20211 (referred to as culture medium 1) and culture media of *Pectinobacterium diversicolor* FN20111 (referred to as culture medium 2).

[0065] After activating strain G15, single colonies were picked and cultured in LB liquid medium at 37°C with shaking at 200 rpm until OD500 was reached. 600 Value = 1.0, yielding bacterial suspension of strain G15.

[0066] Initial screening process: Take 50 μL of culture medium 1 and spread it evenly on LB medium plate. After standing for 5 minutes, place 5 filter paper discs and add 10 μL of Bacillus pumilus G15 to the filter paper discs. After drying, incubate at 37℃ upside down for 1 to 2 days and then observe the results.

[0067] Set up a water control group-1: Take 50 μL of culture medium 1 and spread it evenly on an LB medium plate. After standing for 5 minutes, place 5 filter paper discs, take 10 μL of water and add it to the filter paper discs. After drying, incubate at 37℃ upside down for 1 to 2 days and then observe the results.

[0068] Take 50 μL of culture medium 2 and spread it evenly on an LB medium plate. After standing for 5 minutes, place 5 filter paper discs on the plate. Take 10 μL of Bacillus pumilus G15 and add it to the filter paper discs. After drying, incubate at 37°C upside down for 1-2 days and then observe the results.

[0069] Set up a water control group-2: Take 50 μL of culture medium 2 and spread it evenly on an LB medium plate. After standing for 5 minutes, place 5 filter paper discs, take 10 μL of water and add it to the filter paper discs. After drying, incubate at 37℃ upside down for 1 to 2 days and then observe the results.

[0070] The secondary screening process is the same as the primary screening process, except that instead of placing 5 filter paper discs in each dish, only 1 filter paper disc is placed in the center of each dish. The secondary screening results are shown below. Figure 2 .

[0071] Plate inhibition tests revealed that other strains isolated in Example 1, such as Actinomycetes, Trichoderma, and Pseudomonas, did not exhibit antagonistic effects against Pectinobacter. Only strain G15 showed antibacterial activity. Figure 2 It can be seen that strain G15 has a good inhibitory effect on *Pectobacterium diversicolor* FN20111, with an inhibition zone diameter of 19.1±0.9 mm (see...). Figure 2 Strain G15 had no inhibitory effect on P. parvum FN20211.

[0072] Figure 2 In the middle: the left side shows the inhibitory effect of G15 on various pectinobacteria FN20111; the right side shows the water control treatment.

[0073] 3. Molecular biological identification of biocontrol strains

[0074] (1) Strain G15 was identified as Bacillus pumilus (abbreviated as B. pumilus).

[0075] The specific identification process is as follows: This invention downloads the genome information of 12 Bacillus strains that have been sequenced from the NCBI database and performs gene similarity analysis on them.

[0076] Average nucleotide identity (ANI) refers to the similarity of homologous genes between two genomes. Generally, an ANI value above 95% indicates the same species. Strain G15 has the highest ANI value of 99.49% with *Bacillus pumilus*, and its ANI values ​​with several other *Bacillus pumilus* strains are all greater than 95%; however, its ANI values ​​with several other closely related *Bacillus* strains are all less than 95%. A DDH value of 70% is generally considered the threshold for different species. The DDH values ​​of strain G15 with several other *Bacillus pumilus* strains are all above 70%, indicating that strain G15 is most closely related to *Bacillus pumilus*. Furthermore, phylogenetic trees were constructed using iqtree from the whole genome sequences of representative strains of each *Bacillus* species, and strain G15 clustered with *B. pumilus*. In conclusion, strain G15 was identified as *B. pumilus* (see Table 1 and [other tables]). Figure 3 .

[0077] Table 1. Average Nucleotide Identification (ANI) and DNA Homology (DDH) values ​​of closely related species of Bacillus.

[0078]

[0079]

[0080] (2) Molecular biological identification of Pectinobacterium

[0081] Based on the alignment results of sequencing using the universal primers gapA-F and gapA-R for Pectinobacter, a phylogenetic tree was constructed using NCBI. Figure 4 FN20211 was identified as belonging to P. parvum, and FN20111 belonged to P. versatile. Figure 4Phylogenetic tree constructed based on the gapA gene sequences of strains FN20211 and FN20111.

[0082] 4. Determination of the efficacy of biocontrol strains against aerial stem rot

[0083] (1) Pathogenicity determination of pectinobacter and Bacillus on potato tubers

[0084] To clarify the virulence of two pectinobacteria and a Bacillus species that inhibits their growth on potato tubers, *Pectinobacterium diversicolor* FN20111 and *Bacillus pumilus* G15 were selected for pathogenicity testing on potato tubers. The specific procedure is as follows:

[0085] After activating *P. vervainii* FN20111 by streaking on LB agar plates, the culture was incubated at 28°C for 24 h to obtain single colonies. These single colonies were then picked and cultured in LB liquid medium at 28°C with shaking at 200 rpm for 12–18 h. The resulting culture was centrifuged at 8500 rpm for 1 min to collect the cells. The cells were washed with 10 mM NaCl and resuspended to obtain a resuspension of *P. vervainii* FN20111 (denoted as FN20111-resuspension 1).

[0086] After activating Bacillus pumilus G15 by streaking on LB agar plates, the culture was incubated at 37°C for 24 h to obtain single colonies of Bacillus pumilus G15. The obtained single colonies of Bacillus pumilus G15 were picked and cultured in LB liquid medium at 37°C with shaking at 200 rpm for 12–18 h. The resulting culture was centrifuged at 8500 rpm for 1 min to collect the bacterial cells. The bacterial cells were washed with 10 mM NaCl and resuspended to obtain a resuspension of Bacillus pumilus G15 (denoted as G15-resuspension 2).

[0087] Select potato tubers (Wotu No. 5) of uniform size that are highly susceptible to aerial stem rot, wash them, disinfect them with sodium hypochlorite for 20 minutes, rinse them thoroughly with sterile water, and air dry them. Use a sterile yellow nozzle to puncture the potato tubers to create cavities, then add 50 μL of FN20111-resuspended solution 1 and 50 μL of G15-resuspended solution 2 to the cavities, respectively, and set up 5 parallel experiments.

[0088] A buffer solution (10 mM NaCl) was added to the cavity of a potato as a negative control.

[0089] Afterwards, each group sealed the wound with Vaseline, wrapped the potato tubers with paper towels, laid them flat in a food storage box, and sprayed them with sterile water to keep them moist. After incubation in the dark at room temperature for 48–72 hours, the virulence of Pectinobacterium and Bacillus was assessed based on the weight of the soaked tissue. Figure 5To illustrate the virulence of various pectinobacteria and Bacillus pumilus on potato tubers, A shows longitudinal sections of tubers inoculated with various pectinobacteria FN20111 and Bacillus pumilus G15; B shows the difference in internal decay weight of tubers under different treatments.

[0090] The results show that various types of pectinobacterium FN20111 caused varying degrees of rot in potato tubers, while Bacillus pumilus G15 did not cause rot in potato tubers. Figure 5 (A). The weight of the decayed part caused by *Pectobacterium diversicolor* FN20111 was 3.62 ± 0.26 g ( Figure 5 (B) The weight of the decayed part caused by Bacillus pumilus G15 was 0.17 ± 0.03 g ( Figure 5 The results showed no significant difference between the two, indicating that Bacillus pumilus G15 does not cause rot or other symptoms in potato tubers, and an antibacterial test can be conducted on the pathogen.

[0091] (2) Determination of the efficacy of Bacillus subtilis in inhibiting potato aerial stem rot in tubers

[0092] To clarify whether biocontrol Bacillus has a preventive or curative effect against Pectinobacterium on potato tubers, two treatment groups were set up to determine the control efficacy of Bacillus on potato aerial stem rot pathogens on tubers. Specifically:

[0093] Treatment 1: Select potato tubers (Wotu No. 5) of uniform size that are highly susceptible to aerial stem rot. After washing, disinfect with sodium hypochlorite for 20 minutes, rinse thoroughly with sterile water, and air dry. Use a sterile yellow nozzle to puncture the potato tubers to create cavities. Add 50 μL of LFN20111-resuspended solution 1 to the cavities, incubate in the dark at 28℃ for 12 hours, and then inoculate with 50 μL of G15-resuspended solution 2. Set up 5 parallel experiments.

[0094] Treatment 2: Select potato tubers (Wotu No. 5) of uniform size that are highly susceptible to aerial stem rot, wash them, disinfect them with sodium hypochlorite for 20 minutes, rinse them thoroughly with sterile water, and air dry them. Use a sterile yellow nozzle to puncture the potato tubers to create cavities, then add 50 μL of G15-resuspended solution 2 into the cavities. After incubating in the dark at 28°C for 12 hours, inoculate with 50 μL of FN20111-resuspended solution 1. Set up 5 parallel experiments.

[0095] See results Figure 6 It can be seen that the weight of the rotten part of the potato tuber caused by inoculation with *Bacillus pumilus* FN20111 for 12 hours followed by inoculation with *Bacillus pumilus* G15 was 3.18 ± 0.29 g. The weight of the rotten part of the potato tuber caused by inoculation with *Bacillus pumilus* G15 for 12 hours followed by inoculation with *Bacillus pumilus* FN20111 was 0.23 ± 0.04 g. Figure 6In contrast, the treatment of inoculating with various types of pectinobacterium FN20111 for 12 hours followed by inoculation with G15 significantly reduced the area of ​​decayed tissue. Figure 6 Therefore, Bacillus pumilus G15 has an inhibitory effect on various pectinobacteria FN20111.

[0096] 5. Test of potted plant efficacy

[0097] To determine the control efficacy of Bacillus pumilus G15 against various pectinobacteria FN20111 in potted potatoes, its efficacy was tested on potted potatoes. The experimental method is as follows:

[0098] (1) Preparation of resuspension: Bacillus pumilus G15 was activated and streaked on LB agar plates, and cultured in a 37°C incubator for 24 h. Single colonies were then picked and placed in LB liquid medium and cultured at 37°C and 200 rpm for 12–14 h to prepare the seed culture. The seed culture was added to LB liquid medium at a volume ratio of 1:100 and cultured with shaking until the OD of the culture medium was reached. 600 =1.0, the obtained culture medium was centrifuged at 8500 rpm for 1 min to collect the bacterial cells, washed with physiological saline and resuspended to 10. 7 CFU / mL was used to obtain G15 resuspension.

[0099] Activated *Pectobacterium diversicolor* FN20111 was streaked onto LB agar plates and incubated at 28°C for 24 hours. Single colonies were then picked and incubated in LB liquid medium at 28°C and 200 rpm for 12–14 hours to prepare a seed culture. The seed culture was added to LB liquid medium at a volume ratio of 1:100 and cultured with shaking until the OD of the culture medium reached [value missing]. 600 =0.8, the obtained culture medium was centrifuged at 8500 rpm for 1 min to collect the bacterial cells, washed with physiological saline and resuspended to 10. 7 CFU / mL was used to obtain FN20111 resuspension.

[0100] (2) Treatment 1: The potato variety Wotu No. 5, which is highly susceptible to aerial stem rot, was selected. The tubers were cut into pieces, wrapped in talcum powder, and planted in pots. Watering was carried out every 7 days. After about 30 days of cultivation, 30 mL of G15 suspension was sprayed on the stem. After 7 days of spraying, it was inoculated with various pectin bacteria FN20111.

[0101] Inoculation with FN20111 resuspension was performed via injection. The specific procedure was as follows: Before injection, the inoculation site was disinfected with 75% ethanol. 30 μL of FN20111 resuspension was drawn into a 1 mL syringe and injected into the base of the potato stem. After injection, the injection wound was sealed with Vaseline to prevent contamination.

[0102] Treatment 2 (negative control): Same as treatment 1, except that both G15 resuspension and FN20111 resuspension were replaced with physiological saline.

[0103] Treatment 3 (positive control): Same as treatment 1, except that FN20111 resuspension was inoculated and Bacillus pumilus G15 resuspension was not sprayed.

[0104] Treatment 4 (drug control): Same as treatment 1, except that the inoculation with Pectinobacterium tumefaciens FN20111 resuspension was replaced with spraying 4% kasugamycin instead of spraying G15 resuspension.

[0105] Ten stems were inoculated for each treatment. The control effect of Bacillus was determined by comparing the length of the soft rot in the longitudinal section of the potato plant stem.

[0106] The inhibitory effect of Bacillus pumilus G15 on various pectinobacteria FN20111 in potato pots is shown in [reference needed]. Figure 7 It can be seen that the average internal decay length of treatment 1, which was sprayed with Bacillus pumilus G15 and then inoculated with Pectinobacterium multiflorum FN20111, was 4.76 ± 0.62 cm. The average internal decay length of treatment 3, which was inoculated only with Pectinobacterium multiflorum FN20111 (positive control), was 10.1 ± 0.51 cm. Figure 7 In treatment B), the average control efficacy of Bacillus pumilus G15 against Pectobacterium diversicolor FN20111 was 52.87%. Treatment 4 with 4% kasugamycin showed an average control efficacy of 71.90% against Pectobacterium diversicolor FN20111.

[0107] In summary, this invention provides a biocontrol Bacillus strain, Bacillus spumulus G15, which inhibits the pathogen causing potato aerial stem rot. Bacillus spumulus G15 of this invention exhibits a significant inhibitory effect on *Pectinobacterium diversicolor* FN20111. This invention also provides a microbial agent. Examples show that spraying a resuspension of Bacillus spumulus G15 onto potted potatoes results in a 52.87% control efficacy against *Pectinobacterium diversicolor* FN20111.

[0108] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus pumilus G15, characterized in that, The Bacillus pumilus G15 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32839.

2. A microbial agent, characterized in that, Includes the Bacillus simulans G15 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The effective viable count of Bacillus pumilus G15 in the bacterial agent is ≥1×10⁻⁶. 7 CFU / mL.

4. The microbial agent according to claim 2, characterized in that, The application forms of the Bacillus pumilus G15 include bacterial suspension and / or fermentation broth.

5. The microbial agent according to claim 4, characterized in that, The preparation of the fermentation broth includes: inoculating the Bacillus pumilus G15 into a culture medium and culturing it to obtain the fermentation broth; The preparation of the bacterial suspension includes: centrifuging and resuspending the fermentation broth to obtain the bacterial suspension.

6. The bacterial agent according to claim 1, Bacillus pumilus G15, or any one of claims 2-5, for the control of various pectinobacteria ( P. versatile Application in pathogenic bacteria.

7. The use of Bacillus pumilus G15 as claimed in claim 1 or the inoculum agent as claimed in any one of claims 2 to 5 in the control of potato diseases caused by P. versatile.

8. The application according to claim 7, characterized in that, The diseases include potato blackleg and / or potato aerial stem rot.

9. A method for controlling potato diseases, characterized in that, The Bacillus pumilus G15 of claim 1 or the inoculum of any one of claims 2 to 5 is applied to potatoes; the potato disease is a potato disease caused by P. versatile.

10. The method according to claim 9, characterized in that, When used in the field, the application rate of the microbial agent is 1.9~2.1L / 667m². 2 When used in potted plants, the amount of the microbial agent sprayed is 28-32 mL per seedling.

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